Novel strain of lactobacillus rhamnosus and its metabolites for use in inhibiting xanthine oxidase and treating gout

ABSTRACT

A method for inhibiting xanthine oxidase and for reducing uric acid levels using a composition obtained by culturing  Lactobacillus rhamnosus  in a medium. Also disclosed is a composition including a metabolite of  Lactobacillus rhamnosus  for reducing uric acid levels in a subject and a method for producing the composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/823,585 filed on Aug. 11, 2015, which claims the benefit of U.S.Provisional Application Ser. No. 62/040,616, which was filed on Aug. 22,2014. The content of both prior applications is hereby incorporated byreference in its entirety.

BACKGROUND

Field of the Invention

The invention relates to inhibition of xanthine oxidase activity bylactic acid bacteria and their fermentation metabolites.

Background Information

Uric acid is the end product of purine metabolism in the body. A highlevel of uric acid in the blood leads to the formation and deposition ofuric acid crystals in the joints, kidneys, and other organs. A blooduric acid concentration higher than 7 mg/dL is considered to behyperuricemia.

Hyperuricemia is a common metabolic disorder that is associated withgout, hypertension, cardiovascular disease, diabetes, and kidneydisease. An epidemiological survey performed in Taiwan from 1993 to 2008indicated that the percentage of male and female patients demonstratinghyperuricemia was 21.6% and 9.57%, respectively.

Xanthine oxidase is a key enzyme in the synthesis of uric acid. As aresult, inhibition of xanthine oxidase activity can reduce theproduction of uric acid. Indeed, the xanthine oxidase inhibitor,uricase, is effective for lowering the concentration of uric acid in theblood. Uricase is an enzyme not found in humans. It is typicallyisolated as a recombinant mammalian protein and administered by IVinfusion. As such, it can be expensive to produce and difficult toadminister.

Allopurinol is also a xanthine oxidase inhibitor. This compound isadministered clinically to lower serum uric acid levels. However,allopurinol has side effects, such as allergic reactions,gastrointestinal discomfort, leukopenia and thrombocytopenia, hepatitis,nephropathy, and 6-mercaptopurine toxicity, which in certain cases canlead to death.

In view of the drawbacks of existing therapies for hyperuricemia, manybiopharmaceutical companies focused on the development of new uricacid-lowering agents. For example, Izumida et al., J. Antibiotics50:916-918, isolated a compound that can lower uric acid levels, namely,hydroxyakalone, from the marine bacterium Agrobacterium aurantiacum.

Other microbial species have also been shown to possess uric-acidlowering capability, including strains of Lactobacillus fermentum,Lactobacillus pentosus, Lactobacillus gasseri, Lactobacillus oris,Bifidobacterium longum, and Saccharomyces cerevisiae. See, e.g., USPatent Application Publications 2010/0316618, 2011/0014168, is and2013/0330299; and European Patent Application Publications 2457576 and1649863.

The need still exists to develop new xanthine oxidase inhibitors fromnatural sources which can be easily produced and safely administered.

SUMMARY

To meet this need, inhibitors of xanthine oxidase produced by lacticacid bacteria are provided.

A method for inhibiting xanthine oxidase is also provided. The methodincludes the steps of culturing Lactobacillus rhamnosus in a medium toform a composition and contacting the xanthine oxidase with thecomposition.

Also disclosed is a method for reducing uric acid levels in a subject.The method includes culturing Lactobacillus rhamnosus in a medium toform a composition and administering the composition to a subject havingelevated uric acid levels. The amount administered is effective forreducing uric acid levels.

Within the scope of the invention is a method for producing acomposition for reducing uric acid levels in a subject. The method iscarried out by inoculating a medium with Lactobacillus rhamnosus andculturing the Lactobacillus rhamnosus in the medium to form acomposition.

Further disclosed is a composition for reducing uric acid levels in asubject. The composition includes a metabolite of Lactobacillusrhamnosus.

The details of one or more embodiments of the invention are set forth inthe description and the examples below. Other features, objects, andadvantages of the invention will be apparent from the detaileddescription of several embodiments and also from the claims. Allpublications and patent documents cited herein are incorporated byreference in their entirety.

DETAILED DESCRIPTION

As mentioned above, a method is disclosed for reducing uric acid levelsin a subject by administering a composition containing Lactobacillusrhamnosus. In a particular embodiment, the Lactobacillus rhamnosus isLactobacillus rhamnosus I21 deposited under Accession No. DSM 28876.

In one embodiment, the subject is hyperuricemic. In another embodiment,the subject suffers from gout.

The method, as set out supra, includes a step of forming a compositionby culturing Lactobacillus rhamnosus in a medium. The medium can be, butis not limited to, de Man-Rogosa-Sharpe (MRS) broth, milk, and juice. Inspecific embodiments, the medium is grape juice, mango juice, or orangejuice. In a particular embodiment, the method includes a step ofremoving the Lactobacillus rhamnosus from the medium after culturing andprior to administering the composition.

The composition described above can be administered topically orsystemically by routes including, but not limited to, intramuscular,intradermal, intravenous, subcutaneous, intraperitoneal, intranasal,oral, mucosal, and external.

Depending upon the route of administration, the composition can beformulated in various ways. For example, the composition can be a liquidsolution, a suspension, an emulsion, a syrup, a tablet, a pill, acapsule, a sustained release formulation, a powder, a granule, anampoule, an injection, an infusion, a kit, an ointment, a lotion, aliniment, a cream, or a combination thereof. The composition can besterilized or mixed with a pharmaceutically acceptable carrier orexcipient.

The term “carrier” or “excipient” as used herein refers to anysubstance, not itself a therapeutic agent, used as a carrier, diluent,adjuvant, or vehicle (i) for delivery of a therapeutic agent to asubject, (ii) for adding to a formulation to improve its handling orstorage properties, and/or (iii) to facilitate formation of a dosageunit of the composition into a discrete article such as a capsule ortablet suitable for oral administration.

Suitable carriers or excipients are well known in the art ofmanufacturing pharmaceutical formulations or food products. Carriers orexcipients can include, by way of illustration and not limitation,buffers, diluents, disintegrants, binding agents, adhesives, wettingagents, polymers, lubricants, glidants, substances added to mask orcounteract a disagreeable taste or odor, flavors, dyes, fragrances, andsubstances added to improve appearance of the composition.

Acceptable carriers or excipients include citrate buffer, phosphatebuffer, acetate buffer, bicarbonate buffer, stearic acid, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulfuric acids, magnesium carbonate, talc, gelatin, acacia gum, sodiumalginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose,starches, cellulosic materials (e.g., cellulose esters of alkanoic acidsand cellulose alkyl esters), low melting wax cocoa butter, amino acids,urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serumalbumin), ethylenediamine tetraacetic acid (EDTA), dimethyl sulfoxide(DMSO), sodium chloride or other salts, liposomes, mannitol, sorbitol,glycerol or powder, polymers (e.g., polyvinyl-pyrrolidone, polyvinylalcohol, and polyethylene glycols), and other pharmaceuticallyacceptable materials. The carrier does not destroy the pharmacologicalactivity of the therapeutic agent and is non-toxic when administered indoses sufficient to deliver a therapeutic amount of the agent.

The amount of the composition administered is effective for reducinguric acid levels in the subject. A skilled artisan can easily determinethe effective amount by, e.g., measuring changes in the concentration ofuric acid in the blood of the subject.

The method for inhibiting xanthine oxidase described above includes thestep culturing Lactobacillus rhamnosus in a medium to form acomposition. In a preferred embodiment, the Lactobacillus rhamnosus isLactobacillus rhamnosus I21 deposited under Accession No. DSM 28876.

The medium can be, but is not limited to, de Man-Rogosa-Sharpe (MRS)broth, milk, and juice. In specific embodiments, the medium is grapejuice, mango juice, or orange juice. In a particular embodiment, themethod includes a step of lyophilizing the composition to form a powder.

The method for inhibiting xanthine oxidase also includes a step ofcontacting the xanthine oxidase with the composition described above. Inone embodiment, the contacting step can be performed in vitro. Forexample, a preparation of xanthine oxidase can be placed in a vesseltogether with the composition. In an embodiment, the contacting step isperformed by administering the composition orally to a subject havingxanthine oxidase.

Summarized above is a method for producing a composition for reducinguric acid levels in a subject. The composition is produced by firstinoculating a medium with Lactobacillus rhamnosus. In a specificembodiment, the Lactobacillus rhamnosus is Lactobacillus rhamnosus I21deposited under Accession No. DSM 28876.

The medium for inoculating the Lactobacillus rhamnosus can be, but isnot limited to, de Man-Rogosa-Sharpe (MRS) broth, milk, and juice. Incertain embodiments, the medium is grape juice, mango juice, or orangejuice.

After inoculating the medium with Lactobacillus rhamnosus, theinoculated media is subjected to culturing, thereby forming thecomposition for reducing uric acid levels in a subject. The culturingstep can be carried out at 37° C. Additionally, the culturing step canbe carried out under facultative anaerobic conditions. In an embodiment,the culturing is performed for 2 days.

The composition obtained by culturing Lactobacillus rhamnosus in amedium can be sterilized by methods including but not limited topasteurization, irradiation, autoclave, and filtration. For example, thecomposition can be sterilized by filtration through a 0.2 μm filter. Ina particularly preferred embodiment, the sterilized liquid broth isfirst filtered or centrifuged to remove the bacteria and thenconcentrated.

The method for producing a composition for reducing uric acid levels ina subject can include a step of removing the Lactobacillus rhamnosusfrom the composition. The Lactobacillus rhamnosus can have a celldensity of 1×10⁸ to 1×10⁹ cells/ml prior to the removing step. In apreferred embodiment, the Lactobacillus rhamnosus cell density prior toremoving them is 1×10⁹ cells/ml.

In another embodiment, the method includes a step of lyophilizing thecomposition to form a powder.

The composition described above for reducing uric acid levels in asubject includes a metabolite of Lactobacillus rhamnosus. The metaboliteis an inhibitor of xanthine oxidase activity. In a particularembodiment, the metabolite can be a metabolite of Lactobacillusrhamnosus strain I21 deposited under Accession No. DSM 28876.

In an embodiment, the composition can be a Lactobacillus rhamnosuspowder. In an alternative embodiment, the composition is free ofLactobacillus rhamnosus.

In another embodiment, the composition can include, in addition to themetabolite is of Lactobacillus rhamnosus, probiotic microorganismsincluding but not limited to Lactobacillus spp., Bifidobacterium spp.,and Saccharomyces spp. For example, one or more of Lactobacillusfermentum, Lactobacillus pentosus, Lactobacillus gasseri, Lactobacillusoris, Bifidobacterium longum, and Saccharomyces cerevisiae can beincluded in the composition.

The composition can also contain one or more food ingredients, e.g., acolorant, an acidity regulator, an anticaking agent, an antioxidant, abulking agent, a carrier, an emulsifier, a flavor enhancer, a glazingagent, a preservative, a stabilizer, a sweetener, a thickener, anutrient additive, and a flavoring agent.

In yet another embodiment, as mentioned above, the composition includesa pharmaceutically acceptable excipient.

The composition can also be a food product. For example, the compositioncan be a yogurt, a beverage, an ice cream, or a cheese.

Without further elaboration, it is believed that one skilled in the artcan, based on the disclosure herein, utilize the present invention toits fullest extent. The following specific examples are, therefore, tobe construed as merely descriptive, and not limitative of the remainderof the disclosure in any way whatsoever.

EXAMPLES Example 1 Lactic Acid Bacteria Produce a Xanthine OxidaseInhibitory Activity

Thirty four lactic acid bacteria strains were separately inoculated ontoDe Man, Rogosa, and Sharpe (MRS) plates and cultured at 37° C. for 3days. Bacteria strains were isolated from healthy infant feces, bovinefeces, milk solids, bacon, fermented bean curd, botanical garden soil,pickles, and sauerkraut. Bacteria were scraped from each plate using a 1μl sterile inoculation loop, inoculated into 10 ml of MRS broth, andincubated at 37° C. under facultative anaerobic conditions for 1 day toprepare an inoculum. The inoculum was then added to MRS broth at 1%(v/v) and incubated for 1 day at 37° C. under facultative anaerobicconditions. The culture medium was centrifuged and the supernatantcollected for the analysis of xanthine oxidase inhibitory activity.

Xanthine oxidase inhibitory activity was measured as follows. First, 10μl of culture media from each strain was added to a well in a 96-wellplate. Then, 150 μl of 50 mM phosphate-buffered saline (PBS) and 80 μlof 150 μM xanthine was added to each well. An initial absorbance valueat 290 nm (OD_(before)) was determined before adding 10 μl of xanthineoxidase (0.1 U) into each well. After incubating the plate at

${{XOI}\mspace{14mu} (\%)} = \frac{100 \times \left\lbrack {1 - \left( {{OD}_{after} - {OD}_{before}} \right)} \right\rbrack}{\left( {{{Blank}\mspace{14mu} {OD}_{after}} - {{Blank}\mspace{14mu} {OD}_{before}}} \right)}$

25° C. for 30 min., the absorbance value was measured again at 290 nm(OD_(after)). The xanthine oxidase inhibitory activity (XOI) of eachsample was calculated according to the following formula:

The results are shown in Table 1 below. Among the 34 lactic acidbacteria strains tested, two strains, namely, strains I21 and F73 (shownin italics), inhibited xanthine oxidase activity more than 40%.

TABLE 1 Xanthine oxidase inhibitory activity of lactic acid bacteriastrains strain E021 E027 E032 E100 E103 E106 E108 E109 E111 E112 % inh.  8.0 ^(a) 20.6 17.4 7.2 26.2 25.7 30.0 8.7 27.8 32.3 strain I01 I02 I03I04 I07 I08 I10 I11 I15 I16 % inh. 17.6 10.6 7.0 1.4 33.6 20.3 29.4 2.613.9 2.1 strain I18 I21 I28 I29 I30 I32 S10-V1 S16-6 S16-9 S16-10 % inh. 4.4 44.5 18.0 7.3 3.1 2.5 24.3 26.2 32.3 22.8 strain S17-2 F73 13-2En3721 % inh. 29.7 68.1 24.7 18.6 ^(a) values are expressed aspercentage inhibition of xanthine oxidase activity

Example 2 HPLC Analysis of Xanthine Oxidase Activity Inhibition

Lactic acid bacteria strains F73 and I21 were inoculated onto MRS platesand cultured at 37° C. for 3 days. The bacteria were scraped from theplate with a 1 μl sterile inoculation loop, inoculated into MRS broth,and incubated at 37° C. for 1 day to prepare an inoculum. The inoculumwas then added to MRS broth and incubated at 37° C. for up to 7 days.Samples were removed from the culture at day 1, day 2 and day 7,centrifuged, and the supernatant collected for the analysis of xanthineoxidase inhibitory activity.

In a reaction tube, 880 μl of xanthine (50 μg/ml in 100 mM PBS) and 40μl of 50 mM PBS or 40 μl of the culture supernatants were premixed, and80 μl of xanthine oxidase (0.1 U) was added to initiate the reaction.The reaction was incubated at 30° C. for 30 min., after which an equalvolume of absolute ethanol was added to terminate the reaction. Theterminated reaction was filtered through a 0.25 μm membrane filter andthe content of xanthine was analyzed by HPLC. Xanthine oxidaseinhibitory activity of the samples was calculated as follows:

${{XOI}\mspace{14mu} (\%)} = \frac{{100 \times \lbrack{xanthine}\rbrack_{initial}} - \lbrack{xanthine}\rbrack_{{after}\mspace{14mu} {sample}}}{\lbrack{xanthine}\rbrack_{initial} - \lbrack{xanthine}\rbrack_{{after}\mspace{14mu} {control}}}$

The results are shown in Table 2 below.

TABLE 2 Inhibition of xanthine oxidase activity strain 1 day ^(a) 2 days7 days I21 26.74 ^(b) 27.39 24.63 F73 14.95 17.23 23.55 ^(a) number ofdays in culture at which sample was removed ^(b) values expressed aspercentage inhibition of xanthine oxidase activity

The results demonstrated that xanthine oxidase inhibitory activity oflactic acid bacteria strain I21 is higher than strain F73. Notably, thexanthine oxidase inhibitory activity of strain I21 reached a maximumafter 1 day of fermentation. Prolonged culturing of strain I21 for up to7 days did not result in an increase of xanthine oxidase inhibitoryactivity.

Example 3 Identification of Lactic Acid Bacteria Strain I21

Lactic acid bacteria strain I21 was isolated from the feces of a healthyinfant. An analysis of this strain revealed that it was Gram-positive,catalase and oxidase negative, and non-motile. Additionally, the straindid not produce endospores and did grow under both aerobic andfacultative anaerobic conditions.

The sequence of 16S rDNA from strain I21 (SEQ ID NO: 1) was analyzed anddetermined to be most similar to Lactobacillus casei, Lactobacillusparacasei subsp. paracasei, Lactobacillus paracasei subsp. tolerans,Lactobacillus rhamnosus, and Lactobacillus zeae. The 16s rDNA sequencesimilarity is as high as 98%.

An analysis of a partial sequence of the DnaK gene (SEQ ID NO: 2)revealed that strain I21 shares 99% sequence identity to Lactobacillusrhamnosus.

Strain I21 was also characterized with respect to the ability to fermentcertain carbohydrates using the analytical profile index API®identification system. This test revealed that strain I21 is a strain ofLactobacillus rhamnosus.

Applicants deposited Lactobacillus rhamnosus strain I21 on Jun. 2, 2014under the terms of the Budapest Treaty with the International StrainDepositary Leibniz Institute DSMZ-German Collection of Microorganismsand Cell Culture, Inhoffenstr. 7 B, D-38124 Braunschweig GERMANY. Thestrain was assigned Accession No. DSM 28876.

Example 4 Treatment of Experimental Uricemia

Lactobacillus rhamnosus I21 was inoculated on an MRS plate and culturedat 37° C. for 3 days. Bacteria were scraped from the plate using a 1 μlsterile inoculation loop, inoculated into MRS broth, and grown at 37° C.for 1 day to prepare an inoculum. The inoculum (30 ml) was then addedinto 3 L MRS broth in a 5 L fermenter and grown at 37° C. for 2 days.The fermentation broth was centrifuged at 3000 rpm for 15 min. Thesupernatant was collected and lyophilized to produce the Lactobacillusrhamnosus I21 fermentation product.

ICR mice were used as experimental animals. Potassium oxonate, a uricaseinhibitor, was used to induce a high level of uric acid in the serum ofthe mice. Mice were fasted for one hour and then fed saline or potassiumoxonate (400 mg/kg) via a feeding tube. After one hour, potassiumoxonate-treated mice were fed saline, allopurinol (10 mg/kg), or aLactobacillus rhamnosus I21 fermentation product (150 mg or 200 mgresuspended in saline per mouse) prepared as described above. Tenanimals were used for each experimental group and for the control group.The animals were sacrificed after one hour and the level of uric acid intheir serum was analyzed. The results are shown in Table 3 below.

TABLE 3 A fermentation product of Lactobacillus rhamnosus I21 can reduceserum uric acid levels in experimental animals. serum uric acidExperimental group ^(a) concentration saline control 3.51 ± 0.02 mg/dLpotassium oxonate (400 mg/kg) 4.91 ± 0.08 mg/dL potassium oxonate +allopurinol (10 mg/kg) 2.82 ± 0.28 mg/dL potassium oxonate + 150 mgfermentation product 4.00 ± 0.49 mg/dL potassium oxonate + 200 mgfermentation product 3.86 ± 0.13 mg/dL ^(a) mice (N = 10 per condition)fed saline or the compounds indicated in a total volume of 200 μl

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, a person skilled in the art can easilyascertain the essential characteristics of the present invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the present invention to adapt it tovarious usages and conditions. Thus, other embodiments are also withinthe claims.

1. A composition for reducing uric acid levels in a subject, thecomposition comprising a metabolite of Lactobacillus rhamnosus mixedwith a pharmaceutically acceptable carrier selected from the groupconsisting of ethylenediamine tetraacetic acid, dimethyl sulfoxide,polyvinyl-pyrrolidone, polyvinyl alcohol, polyethylene glycol, and acombination thereof, wherein the metabolite is an inhibitor of xanthineoxidase activity and the Lactobacillus rhamnosus is Lactobacillusrhamnosus strain I21 deposited under Accession No. DSM
 28876. 2. Thecomposition of claim 1, wherein the composition is free of Lactobacillusrhamnosus.
 3. The composition of claim 2, wherein the composition issterile.
 4. The composition of claim 1, wherein the composition issterile.
 5. The composition of claim 1, further comprising a probioticmicroorganism selected from the group consisting of Lactobacillus spp.,Bifidobacterium spp., Saccharomyces spp, and a combination thereof. 6.The composition of claim 5, wherein the probiotic microorganism is oneor more of Lactobacillus fermentum, Lactobacillus pentosus,Lactobacillus gasseri, Lactobacillus oris, Bifidobacterium longum, andSaccharomyces cerevisiae.
 7. The composition of claim 2, furthercomprising a probiotic microorganism selected from the group consistingof Lactobacillus spp., Bifidobacterium spp., Saccharomyces spp, and acombination thereof.
 8. The composition of claim 7, wherein theprobiotic microorganism is one or more of Lactobacillus fermentum,Lactobacillus pentosus, Lactobacillus gasseri, Lactobacillus oris,Bifidobacterium longum, and Saccharomyces cerevisiae.
 9. The compositionof claim 1, wherein the composition is a liquid solution, a suspension,an emulsion, a syrup, a tablet, a pill, a capsule, a sustained releaseformulation, a powder, a granule, an ointment, a lotion, a liniment, ora cream.
 10. The composition of claim 2, wherein the composition is aliquid solution, a suspension, an emulsion, a syrup, a tablet, a pill, acapsule, a sustained release formulation, a powder, a granule, anointment, a lotion, a liniment, or a cream.
 11. A composition forreducing uric acid levels in a subject, the composition comprising aculture of Lactobacillus rhamnosus mixed with a pharmaceutically isacceptable carrier selected from the group consisting of ethylenediaminetetraacetic acid, dimethyl sulfoxide, polyvinyl-pyrrolidone, polyvinylalcohol, polyethylene glycol, and a combination thereof, wherein thecomposition is an inhibitor of xanthine oxidase activity and theLactobacillus rhamnosus is Lactobacillus rhamnosus strain I21 depositedunder Accession No. DSM
 28876. 12. The composition of claim 11, whereinthe composition is a liquid solution, a suspension, an emulsion, asyrup, a tablet, a pill, a capsule, a sustained release formulation, apowder, a granule, an ointment, a lotion, a liniment, or a cream. 13.The composition of claim 12, wherein the composition is sterile.
 14. Thecomposition of claim 11, wherein the composition is sterile.
 15. Thecomposition of claim 11, further comprising a probiotic microorganismselected from the group consisting of Lactobacillus spp.,Bifidobacterium spp., Saccharomyces spp, and a combination thereof. 16.The composition of claim 15, wherein the probiotic microorganism is oneor more of Lactobacillus fermentum, Lactobacillus pentosus,Lactobacillus gasseri, Lactobacillus oris, Bifidobacterium longum, andSaccharomyces cerevisiae.
 17. The composition of claim 12, furthercomprising a probiotic io microorganism selected from the groupconsisting of Lactobacillus spp., Bifidobacterium spp., Saccharomycesspp, and a combination thereof.
 18. The composition of claim 17, whereinthe probiotic microorganism is one or more of Lactobacillus fermentum,Lactobacillus pentosus, Lactobacillus gasseri, Lactobacillus oris,Bifidobacterium longum, and Saccharomyces cerevisiae.
 19. Thecomposition of claim 11, wherein the culture of Lactobacillus rhamnosusincludes a culture medium selected from de Man-Rogosa Sharpe broth,milk, and juice.
 20. The composition of claim 11, further comprising oneor more food ingredient selected from the group consisting of acolorant, an acidity regulator, an anticaking agent, an antioxidant, abulking agent, a carrier, an emulsifier, a flavor enhancer, a glazingagent, a preservative, a stabilizer, a sweetener, a thickener, anutrient additive, a flavoring agent, and a combination thereof.